Fig. 5

Off-target analyses for additional NmeCas9 sgRNAs, targeting sites with consensus and variant PAMs. a Number of GUIDE-Seq reads for the on-target sites, with the PAM sequences for each site indicated underneath. b Number of GUIDE-Seq-detected off-target sites using the Bioconductor package GUIDEseq version 1.1.17 [75] with default settings except that PAM.size = 8, PAM = “NNNNGATT,” min.reads = 2, max.mismatch = 6, allowed.mismatch.PAM = 4, PAM.pattern = “NNNNNNNN$,” BSgenomeName = Hsapiens, txdb = TxDb.Hsapiens.UCSC.hg19.knownGene, orgAnn = org.Hs.egSYMBOL gRNA.size was set to length of the gRNA used, and various number of 0’s were added at the beginning of weights to make the length of weights equal to the gRNA size. For example, for gRNA with length 24, weights = c(0,0,0,0,0, 0, 0.014, 0, 0, 0.395, 0.317, 0, 0.389, 0.079, 0.445, 0.508, 0.613, 0.851, 0.732, 0.828, 0.615, 0.804, 0.685, 0.583) for all sixteen sgRNAs used in (a). c Schematic diagrams of NmeCas9 sgRNA/DNA R-loops for the NTS1C (left) and NTS25 (right) sgRNAs, at the GUIDE-Seq-detected on- and off-target sites. Black, DNA residues; boxed nts, PAM; red line, NmeCas9 cleavage site; cyan and purple, mismatch/wobble and complementary nts (respectively) in the NmeCas9 sgRNA guide region; green, NmeCas9 sgRNA repeat nts. d NmeCas9 editing efficiencies at the NTS1C (left) and NTS25 (right) on-target sites, and at the off-target sites detected by GUIDE-Seq from (b), as measured by PCR and high-throughput sequencing. Data are mean values ± s.e.m. from three biological replicates performed on different days. e Comparison of NmeCas9 and SpyCas9 biochemical off-target sites using SITE-Seq analysis